Nontypeable Haemophilus influenzae (NTHi) is a common cause of infection of the respiratory tract in children and adults. The search for an effective vaccine against this pathogen has focused on components of the outer membrane, and peptidoglycan-associated lipoprotein P6 is among the proposed candidates. This study investigated the immunogenicity of P6 in a rat respiratory model. P6 was purified from two strains of NTHi, one capsule- deficient strain and an H. influenzae type b strain, and assessed for clearance of both homologous and heterologous bacterial strains following mucosal immunization. A protective immune response was determined by enhancement of pulmonary clearance of live bacteria and an increased rate of recruitment of phagocytic cells to the lungs. This was most effective when Peyer's patch immunization was accompanied by an intratracheal (IT) boost. However, the rate of bacterial clearance varied between strains, which suggests some differences in anti-P6 immunological defenses recognizing the expression of the highly conserved P6 lipoprotein on the bacterial surface in some strains. P6-specific antibodies in both serum and bronchoalveolar lavage fluid were cross-reactive and did not differ significantly in strain specificity, demonstrating that difference in clearance was unlikely due to differences in P6-specific antibody levels. Serum homologous and heterologous P6-antibody was bactericidal against NTHi even when enhanced clearance had not been observed. Peyer's patch immunization induced P6-specific CD4+ T- helper cell proliferation in lymphocytes isolated from the mesenteric lymph nodes. An IT boost increased the level of P6-specific antibodies in serum and bronchoalveolar lavage fluid, and P6-specific mesenteric node lymphocyte proliferation. Cells from rats immunized with P6 demonstrated proliferation following stimulation with P6 from nonhomologous strains; however, there was some variation in proliferative responses to P6 from different strains in lymphocytes isolated from animals immunized with killed bacteria. The increase in P6-specific antibodies and T-helper cell responses following an IT boost correlated with an increased rate of recruitment of phagocytic cells and enhanced bacterial clearance of both homologous and heterologous bacteria in the lungs. The data suggests that P6 has the potential to afford protection against pulmonary infection by NTHi following the induction of effective antigen-specific B- and T-cell responses in mucosal tissues.